Sensitive and Specific Method for Rapid Identification of Streptococcus pneumoniae Using Real-Time Fluorescence PCR

Abstract

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, meningitis, and otitis media in the United States (2). While traditional antimicrobial therapy has proven an effective treatment in the past, the emergence of penicillin-and multidrug-resistant strains has resulted in an increasing number of cases of illnesses and fatalities (4, 18). Pneumococcal isolation and identification are complicated by antimicrobial suppression of growth in culture and contamination by normal flora alpha-streptococci. Detection by classical techniques, culture, and serological methods can be time-consuming and indeterminate. Sensitive and specific assays that can be completed quickly in the clinical laboratory are essential for early diagnosis and effective therapy. Molecular assays are inherently valuable because detection can be achieved with enhanced sensitivity and specificity, and detection is not diminished with nonviable organisms.Various molecular methods have been employed to assist investigations (8,11,23). These methods include restriction fragment length polymorphism (RFLP)-based protocols and fingerprinting. PCR-based assays for the detection of S. pneumoniae with primers specific to repetitive regions and genes encoding rRNA (12,14,21), pneumococcal surface adhesion A molecule (22), pneumolysin (20,27,30,37), penicillin-binding protein (5,6,7,24,31), and autolysin (10, 25, 26) have been employed with various degrees of success. Autolysin, encoded by the lytA gene, is required for S. pneumoniae pathogenesis and is a well-characterized virulence marker (1). The lytA gene has been shown to have restricted allelic variation and therefore makes an ideal target for specific identification in clinical and epidemiological studies (9, 35). Sequencing of the lytA locus and high-resolution DNA typing of S. pneumoniae demonstrated that the gene is highly conserved within the species (32, 33), and it has been shown that lytA separates S. pneumoniae from the genotypically similiar species Streptococcus mitis and S. oralis (19,34).Real-time PCR with sequence-specific primers and a fluorescent TaqMan probe allows continuous monitoring of in vitr...

Characteristic Signatures of the lytA Gene Provide a Basis for Rapid and Reliable Diagnosis of Streptococcus pneumoniae Infections

“…In a recent comparative study, Messmer et al reported that PCR amplification of an internal fragment of lytA was the most appropriate approach to the correct identification of S. pneumoniae (26). A similar conclusion had been reached in previous studies (17,25).…”

Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples

“…A TaqMan probe specific to the lytA gene was used to identify cultured S. pneumoniae by real-time PCR (McAvin et al, 2001). Using purified bacterial genomic DNA, the PCR assay was found to be both sensitive and specific, but was not tested on clinical specimens.…”

Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and Description of Streptococcus pseudopneumoniae sp. nov

“…All of these tests are based on the use of Statens Serum Institute Omni serum, a reagent that contains capsular antibodies to all known serotypes of S. pneumoniae. Conflicting reports of sensitivity and specificity exist (6,7,8,12,35,37,40,53,56,63). One of the limitations of the agglutination test kits, as with other immunologic techniques, is that pneumococcal cells without capsules may not react, resulting in false-negative results.…”